Rapid development of contrast agents in the recent years has generated a number of different compositions and formulations, which are useful in contrast-enhanced imaging of organs and tissues of human or animal body as well as in therapeutic treatments thereof.
A class of contrast agents, particularly useful for ultrasound contrast imaging, includes suspensions of gas bubbles of nano- and/or micro-metric size dispersed in an aqueous medium. The gas is typically entrapped or encapsulated in a stabilizing film layer comprising, for instance, emulsifiers, oils, thickeners or sugars. These stabilized gas bubbles (dispersed in a suitable physiological solution) are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, “microspheres”, “microbubbles”, “microcapsules” or “microballoons”, globally referred to here as “gas-filled microvesicles” (or “microvesicles”).
Of particular interest are aqueous suspensions of gas-filled microvesicles where the bubbles of gas are bounded at the gas/liquid interface by a very thin envelope (film) involving a stabilizing amphiphilic material (typically a phospholipid) disposed at the gas to liquid interface. Examples of aqueous suspension of gas-filled microvesicles and preparation thereof are disclosed, for instance, in U.S. Pat. Nos. 5,271,928, 5,445,813, 5,413,774, 5,556,610, 5,597,549, 5,827,504, WO 97/29783 and WO2004/069284.
More recently, attention has been given to so-called “molecular imaging”, where suitable target-specific components are used in the formulation of the contrast agents, for allowing selective contrast-enhanced imaging of organs or tissues. Examples of targeting ligands include, for instance, peptides, proteins, antibodies, aptamers or carbohydrates capable of binding to specific receptors expressed by organs or tissues during pathogenic processes such as, for instance, angiogenesis, inflammation or thrombus formation.
For instance, International Patent applications WO 03/74005, WO 03/084574 and WO 2007/067979 describe suitable peptides which selectively target receptors in vulnerable plaques and tumor specific receptors, such as kinase domain region (KDR) and VEGF (vascular endothelial growth factor)/KDR complex. As described in these patent applications, such peptides are used for formulating target-specific gas-filled microvesicles suitable for binding to KDR or VEGF/KDR complex.
Gas-filled microvesicles are typically prepared by suspending a solid formulation (e.g. in the form of a powdered residue, prepared for instance by freeze-drying) into a physiologically acceptable aqueous solution, in the presence of a physiologically acceptable gas. The obtained suspension of gas-filled microvesicles may then be administered, typically by (intravenous) injection.
As observed by the Applicant, the suspension of the solid formulation in the aqueous solution (also referred to the art as “reconstitution of the dry residue) may represent a critical step of the preparation process of the microvesicles, and many parameters of the suspension step (including for instance the type of isotonic agent and its pH) may affect the characteristics of the microvesicles in the final suspensions.
The Applicant has now found that histidine is particularly useful as pH-adjusting agent for preparing suspensions of peptide-containing gas-filled microvesicles in a carbohydrate-containing physiologically acceptable aqueous solution.